Project Summary: The directed beating of motile cilia is a critical aspect of tissue function in a variety of developmental and physiological contexts including proper neural development, egg migration through the oviduct and mucus clearance in the respiratory tract. The loss of cilia motility results in a wide range of phenotypes including hydrocephaly, infertility, situs inversus, and respiratory dysfunction. We have developed the ciliated epithelium of Xenopus larval skin as a model system to ask: How do ciliated cells generate, maintain and ultimately destroy hundreds of cilia and how do they orient those cilia in an organized way? We have developed numerous light microscopic methods for visualizing specific aspects of ciliated cells in the developing skin of Xenopus embryos. Specific to this application we have implemented the use of LITE sheet microscopy. These methods will allow us to visualize the massive centriole duplication required to generate the approximately 150 basal bodies that nucleate the cilia with significantly improved temporal resolution. Additionally, we can visualize and accurately quantify the cytoskeletal interactions that facilitate the establishment of cilia orientation. Using these methods we will address: (1.) Regulation of cytoskeletal dynamics during the polarization of ciliated epithelia, (2.) The regulation of centriole amplification, and (3.) The transdifferentiation of MCCs. Our results will provide an important link between polarity cues, hydrodynamic forces and the regulation of cytoskeletal dynamics during cellular polarization. Additionally, we will continue our efforts to understand the regulation of centriole biogenesis but expand this work to include the scaling mechanism that regulate centriole number. Finally, we will develop the MCCs of Xenopus as a novel model to understand the molecular regulation of transdifferentiation. While our work is focused on ciliated epithelia, the cell and developmental mechanisms we discover will be broadly applicable. The connection between cytoskeletal dynamics and cell polarity is widely accepted in numerous developmental and disease contexts, and our experiments will likely uncover both MCC specific and more general mechanistic features of this connection. Additionally, defects in centriole duplication highly correlate with late stage cancer progression, indicating an uncoupling of duplication from normal cell cycle progression. The cellular process of transdifferentiation is important during regeneration and cancer progression. Our experiments will provide important developmental control over this process allowing us to uncover novel aspects of coupling transcriptional regulation and autophagocytic recycling.